As is known, the analysis of nucleic acids requires preliminary steps of preparation of a specimen of biological material, amplification of the nucleic material contained therein, and hybridization of individual target strands or reference strands, corresponding to the sequences sought. At the end of these preparatory steps, the specimen may also be examined to check whether the amplification has been carried out correctly.
According to the methodology referred to as “real-time polymerase-chain-reaction” or “PCR”, the DNA is amplified through thermal cycles appropriately selected, and the progress of the amplification is detected and monitored by fluorescence during the entire process.
Various methods and apparatuses for inspection of an optical type are known for this purpose. In particular, the methods and apparatuses of an optical type are frequently based upon the phenomenon of fluorescence. The amplification reactions are conducted so that the nucleic acid strands, contained in a recognition chamber provided in a support, include fluorescent molecules or fluorophores. The support is exposed to a light source having an appropriate spectrum of emission, such as to excite the fluorophores. In turn, the excited fluorophores emit a secondary radiation at a wavelength of emission higher than the peak of the excitation spectrum. The light emitted by the fluorophores is collected and detected by an optical sensor. In order to eliminate the background light radiation, which represents a source of noise, the optical sensor is provided with bandpass filters centered at the wavelength of emission of the fluorophores.
The detection of different substances in the same specimen normally requires the use of distinct fluorophores, which have respective excitation and emission wavelengths. Light sources with different emission spectra are then used in succession, for analyzing the responses in the excitation and emission bands of each fluorophore.
PCR analyzers designed for being used for optical reading of the specimens are described in the documents Nos. US20120170608 and US20130004954. These analyzers are designed to read supports provided with a relatively small number of wells (in particular, six wells) containing the specimens for being analyzed, and each well has relatively large dimensions; i.e., it has a square or round shape with a side or diameter between 3 and 4 mm.
Other biochemical and chemical analyses may be similar, sometimes substituting an antibody for the detection of proteins, or other ligands for the detection of other chemicals. However, many are also amendable to a fluorescence-based analysis.
Known systems present some limitations. In particular, the composition of the surface of the chip typically used for analysis generates undesirable reflections of the optical source used for illuminating the support during fluorescence analysis, generating background noise that raises the detection-sensitivity threshold.
Furthermore, the present applicant has found that the systems described in the documents Nos. US20120170608 and US20130004954 do not enable optical reading of supports of silicon having wells of dimensions smaller than those envisaged by the known art described therein (for example, square wells with a side equal to or smaller than 1 mm). Images, typically low-resolution ones (e.g., 300×200 pixels), made by such systems create difficulty in the discrimination of the individual wells and in selective acquisition of the brightness of the fluorescence emitted by the wells. In conclusion, a reading based upon analysis of the fluorescence of said supports is, in effect, impracticable for values of fluorescence below a certain threshold that are typical of a real-time PCR, antibody detection or otherwise.
Analysis on such small volumes envisages use of high-resolution image-acquisition systems, or a modification of the light-emission sources and an increase of the cost and size of the reading apparatus, which is undesirable as increasing cost.
Thus, what is needed in the art are better devices and methods for the collection and analysis of image date when available from a tiny well or other site.